Folate intake and the risk of oral cavity and pharyngeal cancer:
A pooled analysis within the International Head and Neck
Cancer Epidemiology Consortium
Carlotta Galeone1*, Valeria Edefonti2*, Maria Parpinel3, Emanuele Leoncini4, Keitaro Matsuo5, Renato Talamini6, Andrew F. Olshan7, Jose P. Zevallos8, Deborah M. Winn9, Vijayvel Jayaprakash10, Kirsten Moysich10, Zuo-Feng Zhang11, Hal Morgenstern12,13, Fabio Levi14, Cristina Bosetti1, Karl Kelsey15, Michael McClean16, Stimson Schantz17, Guo-Pei Yu18, Paolo Boffetta19, Yuan-Chin Amy Lee20, Mia Hashibe21, Carlo La Vecchia2* and Stefania Boccia4,22*
1
Department of Epidemiology, IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy
2Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy
3
Department of Medical and Biological Sciences, Unit of Hygiene and Epidemiology, University of Udine, Udine, Italy
4Section of Hygiene, Institute of Public Health, Department of Public Health, Universita Cattolica del Sacro Cuore, Rome, Italy
5
Department of Preventive Medicine, Faculty of Medical Sciences, Kyushu University, Kyushu, Japan
6Unit of Epidemiology and Biostatistics, Aviano Cancer Centre, Aviano, Italy
7
Department of Epidemiology, University of North Carolina School of Public Health, Chapel Hill, NC
8Bobby R. Alford Department of Otolaryngology Head and Neck Surgery, Baylor College of Medicine, University of Texas School of Dentistry at Houston,
Houston, TX
9Division of Cancer Control and Population Sciences, National Cancer Institute, Bethesda, MD
10
Department of Cancer Prevention and Control, Roswell Park Cancer Institute, Buffalo, NY
11Department of Epidemiology, UCLA School of Public Health, Los Angeles, CA
12
Department of Epidemiology, School of Public Health and Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI
13Department of Environmental Health Sciences, School of Public Health and Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI
14
Cancer Epidemiology Unit, Institute for Social and Preventive Medicine (IUMSP), Lausanne University Hospital, Lausanne, Switzerland
15Department of Epidemiology, Brown University, Providence, RI
16
Department of Environmental Health, Boston University School of Public Health, Boston, MA
17Department of Otolaryngology, New York Eye and Ear Infirmary, New York, NY
18
Medical Informatics Center, Peking University, Beijing, China
19The Tisch Cancer Institute and Institute for Translational Epidemiology, Icahan School of Medicine at Mount Sinai, New York, NY
20
Department of Family and Preventive Medicine, University of Utah School of Medicine, Salt Lake City, UT
21Department of Family and Preventive Medicine, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT
22
Area of Systems Approaches and Non Communicable Diseases, IRCCS San Raffaele Pisana, Rome, Italy
Key words:oral cancer, folate intake, diet, epidemiology, risk factor *C.G., V.E., C.L.V. and S.B. contributed equally to this work
Grant sponsor:NIH;Grant numbers:NCI R03CA113157, NIDCR R03DE016611;Grant sponsor:Milan study (2006–2009): Italian Association for Research on Cancer (AIRC);Grant number:10068;Grant sponsor:Italian Ministry of Education;Grant number:PRIN 2009 X8YCBN;Grant sponsor:Italy Multicenter study: Italian Association for Research on Cancer (AIRC), Italian League Against Cancer and Italian Ministry of Research;Grant sponsor:Swiss study: Swiss League against Cancer and the Swiss Research against Cancer/ Oncosuisse;Grant numbers:KFS-700, OCS-1633;Grant sponsor:Boston study: NIH USA;Grant numbers:R01CA078609, R01CA100679; Grant sponsor:Los Angeles Study: NIH USA;Grant numbers:P50CA090388, R01DA011386, R03CA077954, T32CA009142,
U01CA096134, R21ES011667;Grant sponsor:The Alper Research Program for Environmental Genomics of the UCLA Jonsson
Comprehensive Cancer Center;Grant sponsor:MSKCC study: NIH;Grant number:R01CA051845;Grant sponsor:North Carolina (1994– 1997): NIH USA;Grant number:R01CA061188;Grant sponsor:The National Institute of Environmental Health Sciences;Grant number: P30ES010126;Grant sponsor:US Multicenter study: The Intramural Program of the NCI, NIH, USA;Grant sponsor:Japan (2001–2005): Scientific Research grant from the Ministry of Education, Science, Sports, Culture and Technology of Japan;Grant number:17015052;Grant sponsor:The Third-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan; Grant number:H20-002;Grant sponsor:Italian Association for Research on Cancer (AIRC);Grant number:10491-2010/2013;Grant sponsor:Fondazione Veronesi
DOI:10.1002/ijc.29044
History:Received 29 Jan 2014; Accepted 20 May 2014; Online 26 Jun 2014
Correspondence to:Stefania Boccia, Genetic Epidemiology and Public Health Genomics Unit, Section of Hygiene, Institute of Public Health, Universita Cattolica del Sacro Cuore, L.go F. Vito, 1–00168 Rome, Italy, Tel.: 30154396/35001527, Fax: 139-(0)-6-35001522, E-mail: sboccia@rm.unicatt.it
Epidemiology
There are suggestions of an inverse association between folate intake and serum folate levels and the risk of oral cavity and pharyngeal cancers (OPCs), but most studies are limited in sample size, with only few reporting information on the source of dietary folate. Our study aims to investigate the association between folate intake and the risk of OPC within the International Head and Neck Cancer Epidemiology (INHANCE) Consortium. We analyzed pooled individual-level data from ten case–control studies participating in the INHANCE consortium, including 5,127 cases and 13,249 controls. Odds ratios (ORs) and the corre-sponding 95% confidence intervals (CIs) were estimated for the associations between total folate intake (natural, fortification and supplementation) and natural folate only, and OPC risk. We found an inverse association between total folate intake and overall OPC risk (the adjusted OR for the highest vs. the lowest quintile was 0.65, 95% CI: 0.43–0.99), with a stronger associ-ation for oral cavity (OR 5 0.57, 95% CI: 0.43–0.75). A similar inverse associassoci-ation, though somewhat weaker, was observed for folate intake from natural sources only in oral cavity cancer (OR 5 0.64, 95% CI: 0.45–0.91). The highest OPC risk was observed in heavy alcohol drinkers with low folate intake as compared to never/light drinkers with high folate (OR 5 4.05, 95% CI: 3.43–4.79); the attributable proportion (AP) owing to interaction was 11.1% (95% CI: 1.4–20.8%). Lastly, we reported an OR of 2.73 (95% CI:2.34-3.19) for those ever tobacco users with low folate intake, compared with nevere tobacco users and high folate intake (AP of interaction 510.6%, 95% CI: 0.41-20.8%). Our project of a large pool of case–control studies supports a protective effect of total folate intake on OPC risk.
Oral and pharyngeal cancer (OPC) is the seventh most com-mon cancer worldwide, with more than half a million cases and about 300,000 deaths in 2012.1 Tobacco smoking and alcohol consumption are predominant risk factors for OPC although other factors, including the aspects of diet, may affect the risk.2 In particular, a high intake of fruit and vege-tables has been linked with a lower risk of OPC, whereas a poor nutritional status and unbalanced diet have been related to an elevated risk.2–4The association between dietary habits and OPC was investigated in the International Head and
Neck Cancer Epidemiology (INHANCE) Consortium.5
Die-tary habits reflecting high fruit/vegetable and low red meat intake were associated with reduced head and neck cancer risk (per unit score increment, odds ratio [OR] 5 0.90, 95% confidence interval [CI]: 0.84–0.97).
Folate, also known as vitamin B9, is a water-soluble vita-min and is found naturally in green leafy vegetables, cereals, legumes and fruits. In humans, folate plays the fundamental role of providing methyl groups for de novo deoxynucleotide synthesis and for intracellular methylation reactions.6 Only a few case–control studies, however, addressed the effect of folate on OPC, with inconsistent results.7–11Three out of the five studies reported no relationships with risk,8,9,11 whereas two others found an inverse association.7,10 However, all these studies provided data on natural folate intake only. Folate, in fact, can be derived from both plant and animal foods (natural folate), from fortified food products and from supplements (synthetic folate also known as folic acid).
Alcohol intake and tobacco consumption are reported to impair folate levels.12 Alcohol perturbs the folate metabolism by reducing folate absorption, increasing folate excretion or inhibiting methionine synthase,13,14 whereas tobacco con-sumption increases the folate turnover in response to the rapid tissue proliferation or DNA repair in aerodigestive tis-sues among smokers.15,16
As alcohol and tobacco consumption are the major risk factors for OPC, it is worth assessing whether the effect of folate intake on OPC risk is modified by alcohol and tobacco,10,17,18 and whether there is evidence of interaction between variables.
We considered, therefore, the association between folate intake and the risk of OPC in a pooled analysis of case–con-trol studies participating in the INHANCE Consortium, which covers populations from Europe, North America and Japan.
Material and Methods
Studies and participants
The INHANCE Consortium was established in 2004 and to date includes 35 head and neck cancer case–control studies (several of which are multicenter) for a total of 25,478 cases and 37,111 controls (data, version 1.5).19,20 Cases included patients with invasive tumors of the oral cavity, oropharynx, hypopharynx, larynx, oral cavity or pharynx not otherwise specified or overlapping as defined previously.21,22Details on the case–control studies, harmonizing questionnaire data and
What’s new?
Folate is essential to DNA synthesis and repair, suggesting that folate deficiency, in disrupting normal DNA processes, may facilitate the development of certain cancers, including oral and pharyngeal cancer (OPC). The relationship between folate intake and risk of OPC, however, is unclear. In this analysis of data from the International Head and Neck Cancer Epidemiology (INHANCE) Consortium, high levels of folate intake were found to be inversely associated with overall OPC risk. The associa-tion was strongest for cancer of the oral cavity. Risk of OPC was highest among heavy alcohol drinkers with low folate levels.
data pooling methods for the INHANCE consortium have been described previously.19,21All the studies were performed according to the Declaration of Helsinki and were approved by the local ethics committees, according to the legislations at study conduction.
In our analyses, we excluded laryngeal cancer cases and corresponding controls.
All case–control studies in the INHANCE Consortium were eligible for inclusion in our analysis if information on folate intake was available from the corresponding food fre-quency questionnaire (FFQ) for at least 80% of the subjects. Folate and energy intakes were estimated using validated study-specific food composition tables.23–27 The subjects who lacked information or had inconsistent values on folate intake from FFQ were considered as missing. The cases were divided according to the following anatomic sites: (i) oral cavity (including lip, tongue, gum, floor of mouth and hard palate); (ii) oropharynx (including base of tongue, lingual tonsil, soft palate, uvula, tonsil and oropharynx) and hypo-pharynx (including pyriform sinus and hypohypo-pharynx); (iii) oral cavity, pharynx unspecified or overlapping (not other-wise specified, NOS). The main characteristics of the ten eli-gible studies are summarized in Table 1, including 5,127 cases of oral cavity/pharyngeal cancer (1,613 of the oral cav-ity, 2,571 of oropharynx/hypopharynx and 943 of oral cavity/ pharynx NOS) and 13,249 controls.28–37
The estimate of total folate intake was defined in each study and included at least one of the following sources: nat-ural sources of folate, folate-fortified food products and folate supplementation. The study-specific definition of total folate intake represented the most accurate proxy of the real intake of folate in each population considered. In detail, among the ten studies included, six reported folate estimates exclusively from natural sources.28–31,35,37 Two other studies reported folate estimates from natural sources, as well as from other combined sources (i.e., natural food sources, folate-fortified food products and folate supplementation)34,36and two stud-ies reported folate estimates exclusively from natural sources and combined folate supplementation.32,33
Statistical analysis
The main analyses were based on total folate intake, defined as the most complete information on folate intake reported in each of the ten studies. A secondary analysis was based on those studies (eight studies) providing information on the natural sources of dietary folate only.28–31,34–37 For all the analyses, we calculated the study-specific quintiles for folate intake among controls. The study-specific cutoff values are listed in Table 1.
The association between folate intake and OPC risk was assessed by estimating the ORs and the corresponding 95% CIs, using unconditional logistic regression model for each case–control study, adjusted for age (quinquennia, categori-cally), gender, education level (no formal education, less than junior high school, some high school, high-school graduate,
vocational/some college and college graduate/postgraduate), race/ethnicity (non-Hispanic White, Black, Hispanic/Latino, Asian and other), cigarette smoking (never, 1–10, 11–20, 21–30, 31–40, 41–50, >50 pack-years), alcohol drinking (nondrinkers, 0 to <1, 1 to <3, 3 to <5, 5 drinks/day) and total energy intake (continuous).
The pooled effect estimates from all studies were esti-mated with fixed-effects and random-effects logistic regres-sion models.38 We tested for heterogeneity between the study-specific ORs by conducting a likelihood ratio test com-paring a model that included the product terms between each study (other than the reference study) and the variable of interest and a model without product terms, for the risk of oral cavity and pharyngeal cancers combined and for that of each anatomical subsite. We used the random-effects38 esti-mates when heterogeneity was detected (p < 0.10), and the fixed-effects estimates otherwise. We quantified inconsisten-cies across studies and their impact on the analysis by using Cochrane’s Q and the I2statistic.39,40
We also conducted a sensitivity analysis in which each study was excluded one at a time to ensure that the magni-tude of the overall estimates was not dependent on any spe-cific study. Subgroup analyses were also conducted by stratifying the results for total folate intake according to age, gender, geographic region, education level, study design, can-cer subsite, body mass index, tobacco status and alcohol drinking status.
Effect measure modification was evaluated by testing for deviation from a multiplicative interaction model, using the log-likelihood ratio test to compare the fit of logistic models with and without an interaction term. Biological interaction between alcohol, tobacco smoking and total folate intake was estimated using departure from additivity of effects as the cri-terion of interaction as proposed by Rothman.41 To quantify the amount of interaction, the attributable proportion (AP) owing to interaction was calculated as described by Ander-sson et al.42The AP owing to interaction is the proportion of individuals among those exposed to the two interacting fac-tors that is attributable to the interaction per se and it is equal to 0 in the absence of a biological interaction.
Data analyses were conducted using SAS version 9.2 (SAS Institute, Cary, NC) statistical software.
Results
Among the ten studies included, three were conducted in Europe (26% of total cases and 33% of controls), six in North America (65% of total cases and 44% of controls) and one in Japan (9% of total cases and 23% of controls). Three studies were based on cancer registries, whereas the remaining ones were hospital-based case–control studies (Table 1). Table 2 summarizes the characteristics of the study population, which included a total of 13,133 men and 5,233 women (26.7% of cases and 29.2% of all controls were women). In total, more than 78% of cases and 68% of controls were non-Hispanic
Tab le 1. Chara cteristi cs of the ten individual stu dies on OPC 1 from th e INH ANCE Consortium pooled anal ysis and including information on folate in take Study ID Study loc ation Recruitmen t period Sourc e (cases/ controls) Partic ipation rate of case s and controls (% ) Source s o f folate Qui ntile cu toffs of total fo late intake 3 OPC cas es Con trols Natur al food only Supple ments onl y
All sources together
white. Cases were more likely cigarette smokers and alcohol drinkers than controls (Table 2).
The associations between total folate and folate from natu-ral sources only and OPC risk are summarized in Table 3. Considering the ten studies included in the total folate intake analysis, the overall ORs of OPC were 0.78 (95% CI: 0.67– 0.91) for the second quintile, 0.77 (95% CI 0.61–0.96) for the third quintile, 0.72 (95% CI: 0.51–1.01) for the fourth quintile and 0.65 (95% CI: 0.43–0.99) for the fifth quintile compared to the first quintile, with a significant p-value for trend and heterogeneity between the studies. When the results were stratified by anatomic subsite, the ORs for the highest versus the lowest quintile of total folate intake were 0.57 (95% CI: 0.43–0.75) and 0.58 (95% CI: 0.42–0.81) for oral cavity and NOS, respectively, with no evidence of heterogeneity across studies. The OR for the highest versus the lowest quintile of total folate intake was 0.74 (95% CI: 0.42–1.30) for orophar-ynx/hypopharynx combined, with heterogeneity across stud-ies (p 5 0.06).
Considering the eight studies included in the folate intake from natural sources only, the overall ORs of OPC were 0.75 (95% CI: 0.57–1.00) for the second quintile, 0.74 (95% CI: 0.50–1.10) for the third quintile, 0.70 (95% CI: 0.46–1.06) for the fourth quintile and 0.72 (95% CI: 0.46–1.14) for the fifth quintile compared to the first quintile, with heterogeneity across studies (p < 0.01). When the results were stratified by anatomic subsite, the ORs for the highest versus the lowest quintile of natural folate intake were 0.64 (95% CI: 0.45– 0.91), 0.79 (95% CI: 0.44–1.43) and 0.69 (95% CI: 0.36–1.32) for oral cavity, oropharynx/hypopharynx combined and NOS, respectively, with evidence of heterogeneity across the studies for the latter two subsites.
Table 2.Distribution of OPC cases and controls according to the
selected variables1in the ten studies included in the INHANCE
Consortium
OPC cases Controls
n % n % Age (years) <40 237 4.6 739 5.6 40–44 228 4.5 625 4.7 45–49 526 10.3 1,043 7.9 50–54 785 15.3 1,879 14.2 55–59 953 18.6 2,261 17.1 60–64 814 15.9 2,148 16.2 65–69 734 14.3 2,087 15.7 70–74 542 10.5 1,644 12.4 75 308 6.0 821 6.2 p (v2test) <0.0001 Sex Men 3,753 73.3 9,380 70.8 Women 1,369 26.7 3,864 29.2 p (v2test) 0.001 Race/ethnicity Non-Hispanic white 4,006 78.3 9,064 68.6 Black 484 9.5 627 4.8 Hispanic/Latino 122 2.4 308 2.3 Asian 466 9.1 3,166 24.0 Other 37 0.7 48 0.3 p (v2test) <0.0001 Education No formal 235 4.6 716 5.4
Less than junior high school 1,117 21.8 4,088 30.9
Some high school 1,064 20.8 2,003 15.1
High-school graduate 764 14.9 1,638 12.4 Vocational school, some college 1,317 25.7 2,749 20.8 College graduate/ postgraduate 627 12.2 2,046 15.4 p (v2test) <0.0001
Cigarette smoking (pack-years)
Never smokers 919 18.2 5,239 40.2 1–10 356 7.1 1,788 13.7 11–20 406 8.0 1,422 10.9 21–30 583 11.6 1,248 9.6 31–40 633 12.6 1,136 8.6 41–50 594 11.8 778 6.0 >50 1,546 30.7 1,436 11.0 p (v2test) <0.0001
Alcohol intake (drinks/die)
Nondrinkers 646 13.0 3,303 25.6
Table 2. Distribution of OPC cases and controls according to the selected variables in the ten studies included in the INHANCE Con-sortium (Continued)
OPC cases Controls
n % n % >0 to <1 1,143 22.9 4,300 33.4 1 to <3 1,051 21.1 3,035 23.5 3 to <5 710 14.3 1,255 9.7 5 1,425 28.7 1,001 7.8 p (v2test) <0.0001
Body mass index (kg/m2)
<25 2,942 59.4 6,436 48.9
25 2,014 40.6 6,721 51.1
p (v2test)
<0.0001 Total energy intake (kcal/die)
Mean 6 SD 1,584 6 1,232 1,283 6 939
p (t-test) <0.0001
1The sum does not add up to the total because of some missing
values.
The forest plots show the pooled and study-specific OR estimates for the associations between the highest and the lowest quintile of total folate intake, considering all cancer sites combined and separately (Fig. 1). Out of the ten stud-ies, the ORs of OPC were below unity in eight studies (significant in four) and above unity in two studies (nonsignificant).
Table 4 lists the ORs of OPC for the highest versus the lowest quintile of total folate intake according to the selected covariates. There was little evidence of notable effect modifi-cation, except for a stronger inverse association in the hospital-based studies (OR 5 0.52; 95% CI: 0.40–0.69) com-pared to the population-based ones (OR 5 0.80; 95% CI: 0.63–1.01) (for heterogeneity, p 5 0.02).
The analysis of interaction between total folate intake and alcohol reported an OR of 4.05 (95% CI: 3.43–4.79) for heavy drinkers with a low intake of folate, compared to subjects with low alcohol and intermediate/high total folate intake (for interaction, p 5 0.75). Using the estimated ORs listed in
Table 5, the AP owing to interaction is (4.05 2
1.32 2 3.28 1 1)/4.05 5 11.1% (95% CI: 1.4–20.8%). Thus, we estimate that 11.1% of OPC cases occurring among heavy drinkers with low folate intake was attributable to biological interaction (synergy). As for the interaction between tobacco smoking and folates, we reported an OR of 2.73 (95% CI: 2.34–3.19) for those ever tobacco users with a low folate intake, compared to subjects with never tobacco users and intermediate/high total folate intake (for interaction, p 5 0.90). The AP owing to interaction is (2.73 2 1.33 22.11 1 1)/2.73 5 10.6% (95% CI: 0.4–20.8%), suggesting that around 11% of OPC cases occurring among those ever smokers and with low folate levels occurred because of the interaction among the risk factors.
Discussion
This pooled analysis of ten case–control studies including 5,127 OPC cases provided evidence of an inverse association between folate intake and OPC risk. The estimated associa-tion was stronger for oral cavity cancer, with more than 40% risk reduction for the highest quintile of folate intake, than
Figure 1.Study-specific and pooled estimates of OPC (a), oral cavity (b), oropharynx/hypopharynx (c) and NOS (d) cancer for the highest versus the lowest quintile of total folate intake. INHANCE Consortium.
Table 4.Distribution of cases of OPC and controls, and corresponding OR1and 95% CI, for the highest quintile of total folate intake versus the lowest one in strata of selected covariates. INHANCE Consortium
OPC
Cases2n:n Controls2n:n OR (95% CI)
p-Value for heterogeneity between studies Age (years) <55 350:348 810:751 0.69 (0.40–1.20) <0.01 55 659:603 1,615:1,680 0.70 (0.44–1.12) 0.03
p-Value for heterogeneity between strata 0.97
Gender
Men 674:769 1,637:1,820 0.60 (0.37–0.97) 0.03
Women 335:182 788:611 0.80 (0.55–1.16) 0.23
p-Value for heterogeneity between strata 0.36
Geographic region3
Europe 319:233 828:811 0.67 (0.37–1.19) 0.98
North America 577:667 1,010:1,020 0.73 (0.58–0.90) 0.22
Asia 113:51 587:600 0.51 (0.35–0.75) –
p-Value for heterogeneity between strata 0.29
Education
<high school graduate 325:235 898:908 0.57 (0.40–0.80) 0.24
high school graduate 684:716 1,527:1,523 0.71 (0.57–0.87) 0.21
p-Value for heterogeneity between strata 0.28
Study design
Hospital based 551:387 1,663:1,664 0.52 (0.40–0.69) 0.66
Population based 457:564 761:767 0.80 (0.63–1.01) 0.46
p-Value for heterogeneity between strata 0.02
Body mass index (kg/m2)4
<25 638:542 1,222:1,156 0.61 (0.48–0.79) 0.59
25 339:394 1,186:1,262 0.61 (0.33–1.13) 0.03
p-Value for heterogeneity between strata 1.00
Tobacco consumption4,5
Never tobacco users 141:134 834:874 1.05 (0.48–2.28) <0.01
Light tobacco users 129:140 527:592 0.74 (0.48–1.14) 0.94
Heavy tobacco users 696:644 914:813 0.55 (0.43–0.71) 0.47
p-Value for heterogeneity between strata 0.19
Alcohol consumption6
Never drinkers 140:88 670:570 0.51 (0.32–0.82) 0.24
Light drinkers 438:359 1,266:1,300 0.71 (0.35–1.44) 0.08
Heavy drinkers 431:504 489:561 0.59 (0.39–0.90) <0.01
p-Value for heterogeneity between strata 0.74
1
Random-effects estimates were used when heterogeneity was detected, and fixed-effects otherwise. Adjusted for age, sex, race/ethnicity, educa-tion, study, cigarette smoking (pack-years), alcohol intake and total energy intake (as appropriate). The reference category was the lowest quintile of folate intake in each stratum. Calculation of cutoffs for quintile was based on the distribution of controls in each study (study specific).
2Number of subjects in the lowest quintile (I quintile): Number of subjects in the highest quintile (V quintile).
3
Europe included two studies from Italy28,30and from from Switzerland.29North America included six studies.31–36Asia included one study from Japan.37
4The sum does not add up to the total because of some missing values.
5
Light tobacco users were smokers of20 tobacco-years (combination of pack-years of cigarettes and pack-years of cigars/pipe in cigarette
equiva-lent), or subjects only snuffing tobacco. Heavy tobacco users were smokers of >20 tobacco-years or subjects ever chewing tobacco. Light drinkers
were defined as subjects who drank <3 drinks of alcoholic beverages per day and heavy drinkers3 drinks per day.
for oropharynx/hypopharynx. When pooling the eight studies (3,910 OPC cases and 11,805 controls) detailing the intake of natural folate from diet only, however, the inverse association with OPC was no longer significant.
Only a few case–control studies with limited sample sizes were considered on the association between (natural) folate intake estimated from FFQ and OPC risk.7–11 Little or no association was found in three epidemiological studies on this issue conducted in the USA (OR 5 0.7 for the highest vs. lowest level of intake, in both men and women),9 Central
America (OR 5 1.1, 95% CI: 0.6–2.2)11 and Uruguay
(OR 5 1.3, 95% CI: 0.8–2.2).8 Two subsequent case–control studies, one conducted in Italy and Switzerland from 1992 to 199710 and one in Uruguay from 1996 to 2004,7 found an inverse association between folate intake and OPC risk, with ORs, respectively, of 0.53 (95% CI: 0.40–0.69) and 0.49 (95% CI, 0.24–0.98) for the highest versus lowest level of intake. Another Italian study reported lower serum folate levels in patients with head and neck squamous cell carcinoma (mean value, 4.9 ng/mL) compared to control groups of nonsmokers (mean 5 9.7 ng/mL and p < 0.05) and smokers (mean 5 9.1 ng/mL and p < 0.05).43
The results of our study suggest that total folate intake, including fortified food and supplements, is inversely related to OPC risk. Apart from UCLA study, the study-specific defi-nition of total folate intake represented the most accurate proxy of the real intake of folate in each population consid-ered. In fact, these estimates take into account if supplements and/or folate-fortified food products were commonly used in each population during the enrollment study period. The
UCLA Study35 reported the estimates of natural folate only, but it was conducted in a time and in a place where folate fortification in staple foods was mandated (after January 1998) and dietary supplement use was popular. For this rea-son, we performed a sensitivity analysis by excluding that study. The pooled OR for the highest versus the lowest intake of total folate was 0.62 (95% CI: 0.39–0.98) and was similar to the pooled OR when considering all the ten studies (pooled OR 5 0.65; 95% CI: 0.43–0.99).
It was not possible, however, to determine how much of this association was due to natural or synthetic folate, as information on the intake of the two aforementioned sources was detailed only in two studies, with no chance, therefore, of performing any meaningful sensitivity analysis. Interest-ingly, these studies are the only two that reported an OR of >1 for the highest versus the lowest quintile of total folate intake. As information on natural folate intake only was available, we calculated the pooled OR for the highest versus the lowest quintile of this folate source. This was 1.25 (95% CI: 0.86–1.83), and thus not substantially different from the corresponding pooled OR for total folate intake in these two studies, that is 1.21 (95% CI: 0.87–1.68). Even if it is possible that folic acid may exert a different effect than folate in its natural form44and it is known that the bioavailability of folic acid from supplements is higher than the dietary one,45 the few available data did not show important differences in risks between the two sources of folate.
Owing to potential between-countries variations in folate intake, we decided a priori to calculate study-specific quintiles of folate intake. However, we also considered the relationship between OPC and folate intake using absolute cutoffs, based on the distribution of all controls combined. Using this approach, the ORs for subsequent quintiles, as compared to the lowest one, were 0.69, 0.69, 0.65 and 0.63 for all OPC, and the trend in risk was significant. The results were con-sistent for oral cavity and oropharynx.
Mechanistic evidence provides support for an inverse association between folate intake and cancer risk. Folate defi-ciency may increase the risk of various type of cancers, par-ticularly of the gastrointestinal tract,46 through impaired DNA synthesis and disruption of DNA methylation that may lead to protoconcogene activation.47 The folate pathway is led by the 5,10-methylenetetrahydrofolate reductase gene (MTHFR), which converts the 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate, the primary circulating form of folate and a cosubstrate for homocysteine methylation to
methionine.48 A less active form of MTHFR is present
among the subject carriers of the homozygous C677T variant, which is present in 30% of Caucasians.49 The subjects with impaired enzyme activity have reduced folate concentrations, higher serum homocysteine levels, and higher DNA hypome-thylation compared to those carrying the wild-type allele.50 In line with the principle of Mendelian randomization, it is expected that subjects with reduced MTHFR activity are at higher risk of OPC in view of the reduced serum folate levels.
Table 5.ORs1and 95% CIs of OPC according to total folate intake and alcohol and tobacco consumption. INHANCE Consortium
Total folate intake2
Intermediate to high Low
Alcohol consumption3
Never and light drinkers 1(Ref) 1.32 (1.17–1.48) Cases:controls 1,545:6,538 902:3,286 Heavy drinkers 3.28 (2.89–3.73) 4.05 (3.43–4.79) Cases:controls 1,429:1,735 680:800 Tobacco consumption
Never tobacco users 1(Ref) 1.33 (1.09–1.61)
Cases:controls 429:3,059 241:1,414
Ever tobacco users 2.11 (1.84–2.42) 2.73 (2.34–3.19)
Cases:controls 2,471:4,799 1,299:2,435
1Adjusted for age, sex, race/ethnicity, education, study, cigarette
smok-ing (pack-years) and total energy intake.
2Based on the tertiles of intake. Calculation of cutoffs for tertile of total
folate intake was based on the distribution of controls in each study (study specific).
3Light drinkers were defined as subjects who drank <3 drinks of
alco-holic beverages per day and heavy drinkers3 drinks per day.
The distribution of alleles in a population is expected to be unrelated to the confounders that may distort observational epidemiologic studies because of the random assignment of alleles at the time of gamete formation.51 As such, if a func-tional genetic variant such as C677T of the MTHFR is strongly associated with a modifiable exposure (folic acid intake), it can be used to retrieve an unbiased estimate of the association of such exposure (e.g., dietary folate) with a dis-ease (e.g., OPC). Two meta-analyses on the association between MTHFR and OPC have been published so far, with the results showing the absence of an increased risk of cancer among those carrying the unfavorable gene variants which is associated with low serum folate levels.52,53 Taken together, the results of our study and those from the functional genetic variant association studies suggest that although folate intake is, in principle, beneficial toward the risk of OPC, this effect might be differential according to the exact source of folate.
In our study, we reported an additional excess risk of OPC among those with low folate intake who are also heavy drinkers, which is in line with previous findings.10,17,18 It has been reported that alcohol perturbs folate metabolism by reducing folate absorption, increasing folate excretion or inhibiting methionine synthase,14 and hence an additional risk of OPC might be present among heavy drinkers with low folate intake. Additionally, our results suggest the pres-ence of biological interaction between cigarette tobacco smoke and folates, which is in line with previous studies and the biological significance of tobacco in inducing cellular pro-liferation in aerodigestive tissues as a result of the tissue damage.16 Assuming that the relationships studied are causal and based on the definition of biological interaction between two component causes,41,54 our results suggest that more than 11% of OPC cases among heavy alcohol drinkers with a low folate intake, and around 11% of OPC among those ever smokers with low folate intake have arisen because of the synergistic interaction among the two component causes. Taken together, these results have important implications from a public health point of view as they show that by increasing folate intake at the population level, even in the presence of harmful lifestyle behaviors (alcohol and tobacco), a relevant proportion of OPC cancer might be prevented.
Although our study has its strengths, including its very large size, its capacity to explore effect modification by sev-eral characteristics and the stratified analyses according to cancer subsites, it is not without limitations. First, we were unable to dissect the effect of folate on OPC risk according to the intake of supplements or fortified foods. Second, the investigation might be affected by limitations of case–control studies, including recall bias that generally leads to stronger associations between factors and OPC cancer than in cohort
studies. On the other hand, changes in dietary habits after interview could dilute the risks in cohort investigations. Fur-thermore, we were able to adjust for energy intake in all the studies, and thus reducing the effect of possible systematic under- or over-reporting. Selection bias in case–control stud-ies, especially hospital-based studstud-ies, is also a methodological limitation. Therefore, the weaker association observed in population-based studies may be more valid. Nevertheless, hospital-based case–control studies have the advantage over population-based investigations of a higher comparability of information of cases and controls.55
With reference to confounding, we were able to adjust for major recognized risk factors for OPC as well as for total energy intake, but no information was available in the INHANCE data, version 1.5, on HPV, which is a relevant risk factor for oropharyngeal cancer. If anything, however, the inverse association with folate was stronger for other OPC sites.
Conclusions
In conclusion, findings from this large pooled analysis sug-gest that high levels of folate intake may protect against the risk of OPC, after controlling for potential confounding fac-tors, though we cannot rule out selection bias in the hospital-based case–control studies.
Acknowledgements
The authors thank all of the participants who took part in this research for providing them very insightful and constructive comments, which helped improve this manuscript. The INHANCE core data pooling was
supported by NIH grants (NCI R03CA113157 and NIDCR
R03DE016611). The individual studies were supported by the following grants: Milan study (2006–2009): Italian Association for Research on Cancer (AIRC, grant no. 10068) and Italian Ministry of Education (PRIN 2009 X8YCBN). Italy Multicenter study: Italian Association for Research on Cancer (AIRC), Italian League Against Cancer and Italian Ministry of Research. Swiss study: Swiss League against Cancer and the Swiss Research against Cancer/Oncosuisse (KFS-700, OCS-1633). Boston
study: National Institutes of Health (NIH) US (R01CA078609,
R01CA100679). Los Angeles study: National Institute of Health (NIH)
US (P50CA090388, R01DA011386, R03CA077954, T32CA009142,
U01CA096134 and R21ES011667) and the Alper Research Program for Environmental Genomics of the UCLA Jonsson Comprehensive Cancer Center. MSKCC study: NIH (R01CA051845). North Carolina (1994– 1997): National Institutes of Health (NIH) USA (R01CA061188), and in part by a grant from the National Institute of Environmental Health Sci-ences (P30ES010126). US Multicenter study: The Intramural Program of the NCI, NIH, USA. Japan (2001–2005): Scientific Research grant from the Ministry of Education, Science, Sports, Culture and Technology of Japan (17015052) and grant for the Third-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan (H20-002). The work of S.B. was supported by Italian Association for Research on Cancer (AIRC, grant no. 10491-2010/2013). The work of C.G. and E.L. was supported by Fondazione Veronesi.
References
1. Globocan 2012 v1.0. Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. In: Ferlay J, Soerjomataram I, Ervik
M, et al., eds. IARC Cancer Base No. 10. Lyon, France: International Agency for Research on Cancer, 2013.
2. Mouth, pharynx, and larynx. In: World Cancer Research Fund (WCRF) and American Institute for Cancer Research (AICR). Food, nutrition,
physical activity, and the prevention of cancer: a global perspectiveed. Washington, DC: American Institute for Cancer Research, 2007.245–9. 3. La Vecchia C, Franceschi S, Levi F, et al. Diet
and human oral carcinoma in Europe. Eur J Can-cer B Oral Oncol 1993;29B:17–22.
4. Winn DM. Diet and nutrition in the etiology of oral cancer. Am J Clin Nutr 1995;61:437S–45S. 5. Chuang SC, Jenab M, Heck JE, et al. Diet and
the risk of head and neck cancer: a pooled analy-sis in the INHANCE consortium. Cancer Causes Control 2012;23:69–88.
6. Blount BC, Mack MM, Wehr CM, et al. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implica-tions for cancer and neuronal damage. Proc Natl Acad Sci USA 1997;94:3290–5.
7. Aune D, Deneo-Pellegrini H, Ronco AL, et al. Dietary folate intake and the risk of 11 types of cancer: a case-control study in Uruguay. Ann Oncol 2011;22:444–51.
8. De Stefani E, Ronco A, Mendilaharsu M, et al. Diet and risk of cancer of the upper aerodigestive tract—II. Nutrients. Oral Oncol 1999;35:22–6. 9. McLaughlin JK, Gridley G, Block G, et al. Dietary
factors in oral and pharyngeal cancer. J Natl Can-cer Inst 1988;80:1237–43.
10. Pelucchi C, Talamini R, Negri E, et al. Folate intake and risk of oral and pharyngeal cancer. Ann Oncol 2003;14:1677–81.
11. Weinstein SJ, Gridley G, Harty LC, et al. Folate intake, serum homocysteine and methylenetetra-hydrofolate reductase (MTHFR) C677T genotype are not associated with oral cancer risk in Puerto Rico. J Nutr 2002;132:762–7.
12. Bailey LB. Folate status assessment. J Nutr 1990; 120:1508–11.
13. Barak AJ, Beckenhauer HC, Tuma DJ, et al. Effects of prolonged ethanol feeding on methio-nine metabolism in rat liver. Biochem Cell Biol 1987;65:230–3.
14. Mason JB, Choi SW. Effects of alcohol on folate metabolism: implications for carcinogenesis. Alco-hol 2005;35:235–41.
15. Heimburger DC. Localized deficiencies of folic acid in aerodigestive tissues. Ann N Y Acad Sci 1992;669:87–95; discussion 95–6.
16. Piyathilake CJ, Hine RJ, Dasanayake AP, et al. Effect of smoking on folate levels in buccal muco-sal cells. Int J Cancer 1992;52:566–9.
17. Matsuo K, Rossi M, Negri E, et al. Folate, alco-hol, and aldehyde dehydrogenase 2 polymor-phism and the risk of oral and pharyngeal cancer in Japanese. Eur J Cancer Prev 2012;21:193–8. 18. Shanmugham JR, Zavras AI, Rosner BA, et al.
Alcohol-folate interactions in the risk of oral can-cer in women: a prospective cohort study. Cancan-cer Epidemiol Biomarkers Prev 2010;19:2516–24. 19. Conway DI, Hashibe M, Boffetta P, et al.
Enhancing epidemiologic research on head and neck cancer: INHANCE—The international head and neck cancer epidemiology consortium. Oral Oncol 2009;45:743–6.
20. Leoncini E, Ricciardi W, Cadoni G, et al. Adult height and head and neck cancer: a pooled analy-sis within the INHANCE Consortium. Eur J Epi-demiol 2014;29:35–48.
21. Hashibe M, Brennan P, Benhamou S, et al. Alco-hol drinking in never users of tobacco, cigarette
smoking in never drinkers, and the risk of head and neck cancer: pooled analysis in the Interna-tional Head and Neck Cancer Epidemiology Con-sortium. J Natl Cancer Inst 2007;99:777–89. 22. Leoncini E, Ricciardi W, Cadoni G, et al. Adult
height and head and neck cancer: a pooled analy-sis within the INHANCE Consortium. Eur J Epi-demiol 2014;29:35–48.
23. Gnagnarella P, Parpinel M, Salvini S, et al. The update of the Italian Food Composition Database. J Food Compos Anal 2004;17:509–22.
24. Gnagnarella P, Salvini S, Parpinel M. Food Com-position Database for Epidemiological Studies in Italy. Version 2. Milan, Italy: Istituto Europeo di Oncologia, 2008.
25. U.S. Department of Agriculture ARS. USDA National Nutrient Database for Standard Reference, Release 11 and following. In: Laboratory ND, ed. Home Page, http://www.ars. usda.gov/ba/bhnrc/ndl.
26. U.S. Department of Agriculture ARS. USDA. Agriculture Handbooks No.8, 1–19ed. Composition of foods: raw, processed, prepared. USDA National Nutrient Database for Standard Reference. http://www.nal.usda.gov/ref/ USDApubs/aghandbk.htm#sortnbr. 27. Resources Council, Science and Technology
Agency, Japan. Standard tables of food composition in Japan, 5th revised edn. Tokyo: Ministry of Finance Printing Bureau, 2000. 28. Bosetti C, Gallus S, Trichopoulou A, et al.
Influ-ence of the Mediterranean diet on the risk of cancers of the upper aerodigestive tract. Cancer Epidemiol Biomarkers Prev 2003;12:1091–4. 29. Levi F, Pasche C, La Vecchia C, et al. Food
groups and risk of oral and pharyngeal cancer. Int J Cancer 1998;77:705–9.
30. Bravi F, Bosetti C, Filomeno M, et al. Foods, nutrients and the risk of oral and pharyngeal cancer. Br J Cancer 2013;109:2904–10. 31. Jayaprakash V, Rigual NR, Moysich KB, et al.
Chemoprevention of head and neck cancer with aspirin: a case-control study. Arch Otolaryngol Head Neck Surg 2006;132:1231–6.
32. Blot WJ, McLaughlin JK, Winn DM, et al. Smok-ing and drinkSmok-ing in relation to oral and pharyn-geal cancer. Cancer Res 1988;48:3282–7. 33. Schantz SP, Zhang ZF, Spitz MS, et al. Genetic
susceptibility to head and neck cancer: interaction between nutrition and mutagen sensitivity. Laryn-goscope 1997;107:765–81.
34. Peters ES, McClean MD, Liu M, et al. The ADH1C polymorphism modifies the risk of squa-mous cell carcinoma of the head and neck associ-ated with alcohol and tobacco use. Cancer Epidemiol Biomarkers Prev 2005;14:476–82. 35. Cui Y, Morgenstern H, Greenland S, et al.
Poly-morphism of Xeroderma pigmentosum group G and the risk of lung cancer and squamous cell carcinomas of the oropharynx, larynx and esoph-agus. Int J Cancer 2006;118:714–20.
36. Divaris K, Olshan AF, Smith J, et al. Oral health and risk for head and neck squamous cell carci-noma: the Carolina Head and Neck Cancer Study. Cancer Causes Control 2010;21:567–75. 37. Suzuki T, Wakai K, Matsuo K, et al. Effect of
die-tary antioxidants and risk of oral, pharyngeal and laryngeal squamous cell carcinoma according to
smoking and drinking habits. Cancer Sci 2006;97: 760–7.
38. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986;7:177–88. 39. Higgins JP, Thompson SG, Deeks JJ, et al.
Meas-uring inconsistency in meta-analyses. Br Med J 2003;327:557–60.
40. Higgins JP, Thompson SG, Spiegelhalter DJ. A re-evaluation of random-effects meta-analysis. J R Stat Soc Ser A Stat Soc 2009;172:137–59. 41. Rothman KJ. Measuring interactions. In:
Roth-man KJ, ed. Epidemiology: an introduction. New York: Oxford University Press, 2002. 168–90. 42. Andersson T, Alfredsson L, Kallberg H, et al. Calculating measures of biological interaction. Eur J Epidemiol 2005;20:575–9.
43. Almadori G, Bussu F, Galli J, et al. Serum levels of folate, homocysteine, and vitamin B12 in head and neck squamous cell carcinoma and in laryn-geal leukoplakia. Cancer 2005;103:284–92. 44. Lucock M. Folic acid: nutritional biochemistry,
molecular biology, and role in disease processes. Mol Genet Metab 2000;71:121–38.
45. Hannon-Fletcher MP, Armstrong NC, Scott JM, et al. Determining bioavailability of food folates in a controlled intervention study. Am J Clin Nutr 2004;80:911–8.
46. Tio M, Andrici J, Cox MR, et al. Folate intake and the risk of upper gastrointestinal cancers: a systematic review and meta-analysis. J Gastroen-terol Hepatol 2014;29:250–8.
47. Duthie SJ. Folic acid deficiency and cancer: mechanisms of DNA instability. Br Med Bull 1999;55:578–92.
48. Rozen R. Genetic predisposition to hyperhomo-cysteinemia: deficiency of methylenetetrahydrofo-late reductase (MTHFR). Thromb Haemost 1997; 78:523–6.
49. Botto LD, Yang Q. 5,10-Methylenetetrahydrofo-late reductase gene variants and congenital anomalies: a HuGE review. Am J Epidemiol 2000; 151:862–77.
50. Graziano F, Kawakami K, Ruzzo A, et al. Methyl-enetetrahydrofolate reductase 677C/T gene poly-morphism, gastric cancer susceptibility and genomic DNA hypomethylation in an at-risk Ital-ian population. Int J Cancer 2006;118:628–32. 51. Smith GD, Ebrahim S. ‘Mendelian
randomiza-tion’: can genetic epidemiology contribute to understanding environmental determinants of disease? Int J Epidemiol 2003;32:1–22. 52. Boccia S, Boffetta P, Brennan P, et al.
Meta-anal-yses of the methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and risk of head and neck and lung cancer. Cancer Lett 2009;273:55–61.
53. Zhuo X, Ling J, Zhou Y, et al. Polymorphisms of MTHFR C677T and A1298C association with oral carcinoma risk: a meta-analysis. Cancer Invest 2012;30:447–52.
54. Rothman KJ, Greenland S. Causation and causal inference in epidemiology. Am J Public Health 2005;95:S144–50.
55. D’Avanzo B, La Vecchia C, Katsouyanni K, et al. An assessment, and reproducibility of food fre-quency data provided by hospital controls. Eur J Cancer Prev 1997;6:288–93.
